The long-term goal of our research is to understand the molecular mechanisms by which the eukaryotic single-stranded DNA-binding protein, Replication Protein A (RPA), interacts with single-stranded-DNA (ssDNA) and with other proteins in various DNA-processing events. An interaction of RPA with ssDNA is considered to a sequence non-specific. Surprisingly, structural analysis demonstrated that the RPA forms multiple specific hydrogen-bond contacts to cytidine bases in the crystal structure. The aims of the studies described in this proposal are to complete the structural analysis of the remaining ssDNA-binding domains to investigate the DNA-binding induced conformational changes in ssDNA-binding domain and understand a mechanism of sequence non- specificity. To achieve our goals, we will use a combination of biochemical methods and X-ray crystallographic analysis. Specifically our aims are: 1. Does ssDNA binding induce a conformational change in the two ssDNA-binding domains in the largest RPA subunit (RPA70)? The crystal structure of the two ss-DNA binding domains in RPA70 (RPA70/181-422 fragment) will be determined in the absence of ssDNA. 2. What structural features allow the interaction of the two RPA70 ssDNA binding domains with ssDNA to be sequence non-specific? Structures of RPA70181/422 in complex with (dT)8, (dG)8 and (dA)8 will be crystallized and determined to analyze an interaction with each type of DNA base in each position. 3. Is the C-terminal domain of the largest RPA subunit (RPA70) structurally homologues to other ssDNA-binding domains of RPA? What is the mechanism of the RPA trimerisation?. A structure of the C- terminal domain of RPA70 in complex with two smaller subunits, RPA32 and RPA14, will be resolved by X-ray crystallography. 4. What is the general mechanism of ssDNA-binding by the RPA trimer? A trimeric RPA complex containing all ssDNA-binding domains will be resolved by X-ray crystallography with and/or without ssDNA.